Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance

Abstract

Purpose

Salivary duct carcinoma over-expresses epidermal growth factor receptor (EGFR) and HER-2 though underlying mechanisms remain undefined. Because of potential utilization of these markers as treatment targets, we evaluated protein and gene status by several techniques to determine complementary value.

Experimental Design

A tissue microarray of 66 salivary duct carcinomas was used for immunohistochemical analysis of HER-2 and EGFR expression (semiquanititively evaluated into a 3-tiered system), and fluorescence in situ hybridization for gene copy number, and chromosomes 7 and 17 ploidy status. Sequencing of Exons 18, 19 and 21 of the EGFR gene for mutations was performed.

Results

EGFR Forty-six (69.7%) of the 66 tumors showed some form of EGFR expression (17,3+; 17,2+; 12,1+) but none gene amplification. Five (9.4%) of 53 tumors showed mutations in exon 18 (3) and exon 19 (2). Polysomy of chromosome 7(average >2.5 copies per cell) was detected in 15 (25.0%) of 60 tumors (6,3+; 5,2+; 2,1+; 2,0+ expression) and correlated with poor 3-year survival (p=0.015). HER-2: Seventeen (25.8%) of 66 tumors expressed HER-2 (10,3+; 3,2+; 4,1+). Eight tumors showed HER-2 gene amplification (6,3+; 1,1+; 1,0+ protein expression). Chromosome 17 polysomy was found in eight (15.7%) of 51 tumors; two with HER-2 expression (3+; 1+).

Conclusion

Our study shows that salivary duct carcinomas: 1) harbor EGFR gene mutations in a subset of tumors which may guide therapy, 2) pursue aggressive clinical course in cases with Chromosome 7 polysomy and high EGFR expression, and 3) with HER-2 gene amplification and protein high-expression maybe selected for targeted therapy.

Introduction

Salivary duct carcinoma is a high grade clinically aggressive adenocarcinoma variant with remarkable phenotypic and biological resemblance to mammary high grade ductal carcinoma (1, 2). Patients with locally advanced, recurrent and/or metastatic disease have limited therapeutic options and generally succumb to their disease. In the past two decades several individual genes, markers of different cellular pathways, chromosome alterations, and hormonal and growth factor receptors have been evaluated in an effort to define the underlying molecular and biological pathways associated with their development and progression (1, 3-7). Several of these studies have led to the characterization of certain hormonal and growth factors that are known to play a central role in the biology of breast cancers including HER2 and epidermal growth factor receptor (EGFR) (5, 8). However, the optimum methodology that best reflects their functional level for therapeutic proposes remains a subject of debate.

Growth factors and their receptors play a central role in mammary ductal tumorigenesis and in other adenocarcinomas, however, the expression levels alone fail to predict responders to therapies (9). Several studies have shown that HER-2 gene amplification status has improved the identification of possible responders to targeted therapy with trastuzumab (10-14). However, concurrent expression and gene amplification studies have identified a subset of low and intermediate HER-2 gene expressions with high gene amplification suggesting that immunohistochemical (IHC) analysis alone leads to underestimation of patients eligible for therapy (14, 15). Central to addressing this issue, is whether protein expression and/or gene amplification are mutually exclusive or complementary events in assessing the biological role of these factors for therapeutic stratification of patients with these tumors. Accordingly, initial IHC screening followed by gene amplification analysis by fluorescence in situ hybridization (FISH) has been recommended for borderline cases in mammary carcinomas (14, 16). The correlation between the IHC expression and the genomic status of the HER-2 gene in salivary duct carcinoma remains poorly defined secondary to the limited number of tumors studied to date (14, 17-20).

Recently, mutational analysis of the EGFR gene has identified a subset of non-small cell lung carcinomas, that responded to tyrosine kinase inhibitors (TKIs) (21-23). Other molecular alterations including chromosomal polysomy may also play a role in the pathogenesis associated with this pathway (24-28). Since at least half of salivary duct carcinomas express EGFR (5, 29) and little is known on the status of the EGFR expression, correlative assessment of these findings may determine the association between expression and underlying molecular factors including mutational and gene amplification status.

Our objectives were to concurrently analyze, for the first time, the expression and the genomic status of EGFR and HER-2 genes in a large cohort of salivary duct carcinomas. We used immunohistochemical analysis, and fluorescence in situ hybridization to determine their relationship in the biological stratification of patients with salivary duct carcinoma. We also determined the incidence and nature of biomarkers that may guide targeted therapies in a subset of patients with salivary duct carcinoma.

Materials and Methods

Sixty-six salivary duct carcinomas with archival formalin-fixed paraffin blocks available at the University of Texas, M.D. Anderson Cancer Center formed this study. The current World Health Organization guidelines were used for classifying tumors as salivary duct carcinomas (30). A tissue microarray was created using two 1.0 mm diameter cores consisting of representative tumor from each paraffin block consisting of tumor tissue fixed in 10% buffered formalin. The tissue microarray was used for IHC and FISH evaluations. Pathologic findings including IHC, gene amplification, and mutational status were compared with clinical factors including gender, age and stage along with clinical outcomes and were evaluated by Fisher's exact test and Chi Square based on the number of comparative groups. A P-value of 0.05 was considered significant.

Immunohistochemical analysis

Immunohistochemical analysis for HER-2 and EGFR were performed using the automated BOND MAX IHC stainer by Vision Biosystems (Norwell, Massachusetts) on four-micrometer paraffin sections of the tissue microarray material. In brief, following dewaxing, washing and rehydration of the slides through xylene and graded alcohols, Tris-EDTA buffer was used for antigen retrieval. Slides were subsequently treated with 3% hydrogen peroxide to block endogenous peroxidase. Following incubation with the primary antibodies, HER-2 (Clone e2-4001, Mouse, 1:300, Labvision, Fremont, California) and EGFR (Clone 31G7, Mouse, 1:50, Zymed, San Francisco, California) the secondary conjugate antibody was applied. Finally, each specimen-containing slide was developed using the chromogen DAB and counterstained with hematoxylin.

Immunohistochemical stained tumor sections for HER-2 were evaluated for membranous expression: 3+ =strong complete in >10%; 2+= weak complete >10%; 1+ partial staining of tumor cells >10%, 0 =negative or <10% staining. EGFR IHC in tumor cells was evaluated for membranous expression: 3+ =strong membrane staining in >10%; 2+= moderate membranous staining in >10%; 1+ weak membranous staining in >10%, 0 =negative or <10% staining.

Fluorescence in situ hybridization (FISH)

A four micrometer paraffin section of the tissue microarray was analyzed by FISH using the manufacturer's recommended standard methods for HER-2 (PathVysion HER-2 DNA Probe Kit; HER-2 Spectrum Orange/centromere enhancer of position effect (CEP)17 Spectrium Green, Vysis, Abbott, Abbott Park, Illinois) and EGFR (Vysis LSI EGFR Spectrum Orange/CEP 7 Spectrum Green, Vysis, Abbott, Abbott Park, Illinois). A known HER-2 amplified ductal adenocarcinoma was included in the tissue microarray as a positive control as well as normal salivary tissue as a negative control.

Each tumor sample was evaluated for the signals of HER-2 or EGFR signal and amplification pattern (Spectrum Orange) and the number of centromeric probe signals to chromosomes 7 or 17 (Spectrum Green) respectively within at least 20 tumor cells evaluated from both cores. Amplification was defined as clusters of probes (>10 copies per tumor cell) documented in 20% or more of cells analyzed. Tumors were also evaluated for low levels of amplification by comparing the gene to centromeric signals as a ratio, with a ratio <1.8 defined as non-amplified, 1.8 to 2.2 as indeterminate, and a ratio > 2.2 as amplified (14). Additionally, chromosome copy number for Chr 7 and Chr 17 were evaluated. Tumors were classified based on the percentage of cells with increased chromosomal numbers as outlined by Cappuzzo et al. (31) as well as the average number of centromeres per cell with evaluation for correlation with clinical and pathologic parameters. Chromosomal copy numbers per tumor cells were classified as: disomy, up to 2 copies in 90% or more of cells; low trisomy, 3 copies in less than 40% of cells; high trisomy as 3 copies in 40% or more of cells; low polysomy as 4 or more copies in less than 40% of cells; and high polysomy as 4 or more copies in 40% or more of tumor cells (31).

EGFR gene sequence mutational analysis

DNA from paraffin embedded formalin fixed tumor was enriched to >70% tumor cells and was suitable for sequencing Exons 18, 19 and 21, hotspots of the EGFR gene in 53 of the 66 tumors. PCR was performed using previously described primers (27). Amplified target DNA was then sequenced using ABI Big DyeTMv3.1 dye terminator cycle sequencing. Forward and reverse sequencing was performed. A lung adenocarcinoma with known EGFR gene mutation was used as a positive control.

Results

EGFR

a) Immunohistochemistry

Forty-six (69.7%) of 66 salivary duct carcinomas showed membranous expression for the EGFR. Of the positive tumors, 17 (37.0%) showed strong complete membranous staining (3+), 17 (37.0%) were intermediate (2+), and 12 (26.0%) had weak membranous staining (1+) (Table 1).

Table 1. Comparative methods for analyzing EGFR and HER-2 in salivary duct carcinoma.

	IHC- Expression Score
Category	0	1+	2+	3+	Total
EGFR
IHC	20 (30.3%)	12 (18.2%)	17 (25.8%)	17 (25.8%)	66
Gene Amp	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	0 (0.0%)
Polysomy* Chr 7	2/17 (11.8%)	2/12 (16.7%)	5/16 (31.3%)	6/15 (40.0%)	15/60 (25.0%)
HER-2
IHC	49 (74.2%)	4 (6.1%)	3 (4.5%)	10 (15.2%)	66
Gene Amp	1† (2.0%)	1 (25.0%)	0 (0.0%)	6 (60.0%)	8 (12.1%)
Polysomy* Chr 17	6/36 (16.7%)	1/4 (25.0%)	0/3 (0.0%)	1/8 (12.5%)	8/51 (15.7%)

^*Polysomy of corresponding chromosome, average copy number ≥ 2.5.

^†This HER-2 amplified tumor showed 3+ IHC on whole tissue section.

Chr: Chromosome; EGFR: epidermal growth factor receptor; Gene Amp: Gene Amplification; IHC: immunohistochemistry

Although EGFR expression correlated significantly with local recurrence (34.6% vs. 65.4%, p=0.046) and was more frequently associated with distant metastases (44.0% vs. 56.0%) and poor 3 year survival (59.1% vs. 40.9%), it did not reach a statistical significance (p= 0.6 and 0.5, respectively).

b) Gene Amplification(FISH)

EGFR gene amplification was not identified in any of the 66 salivary duct carcinomas analyzed by FISH.

c) Chromosomal Ploidy

An increase in the Chr 7 copy number in tumor cells (average > 2.5 copies per cell), was found in 15 (25.0%) of the 60 tumors suitable for analysis (Table 2, Figure 1B). Tumors with an average ploidy > 2.5 copies for Chr 7 were significantly correlated with a lower three year survival (p=0.002) and difference in overall survival by Kaplan-Meier assessment (p=0.015) (Figure 2). Categorical classification showed 13 (21.7%) tumors with disomy for Chr 7, 25 (41.7%) as low trisomy, 16 (26.7%) as low polysomy, 0 (0.0%) as high trisomy, and 6 (10.0%) as high polysomy (Table 2). Correlative analysis using Kaplan-Meier method showed decline in median survival with increased chromosomal alterations from 52.0 months for Chr 7 disomy to 37.0 months for low trisomy, 30.5 months for low polysomy, and 14.5 months for Chr 7 high polysomy (Table 2, Figure 3). All patients alive without disease were noted to be diploid for Chr 7. Salivary duct carcinomas with increased Chr 7 copy number (> 2.5 average) showed a more rapid clinical course with median survival of 20.0 versus 43.0 months for Chr 7 <2.5 (p=0.015) (Table 2).

Table 2. Assessment of polysomy using average chromosome number versus categorical classification^*.

	Average Chr #		Categorical classification*
Chr	≥2.5	≥3.0	Disomy ≥90%	Low trisomy <40%	High trisomy ≥40%	Low polysomy <40%	High polysomy ≥40%
17 (n=51)	8 (15.7%)	4 (7.8%)	23 (45.1%)	10 (19.6%)	1 (2.0%)	12 (23.5%)	5 (9.8%)
7 (n=60)	15 (25%)	10 (16.7%)	13 (21.7%)	25 (41.7%)	0 (0.0%)	16 (26.7%)	6 (10%)
Median survival†
Chr 7 (mo)	20.0 vs. (43.0 for < 2.5)	22.0 vs. (37.0 for < 3.0)	52	37	na	30.5	14.5

^*Disomy defined as up to 2 chromosome copies in ≥ 90% of tumor cells; low trisomy, 3 chromosomes in up to 40% of cells; high trisomy, 3 chromosomes in ≥ 40% of cells; low polysomy, 4 or more chromosomes in up to 40% of cells; high polysomy, 4 or more chromosomes in ≥ 40% of cells.

^†Median survival from Kaplan Meier Analyses (Figure 2, 3).

Chr, chromosome; mo, months; na, not applicable; vs., versus.

Figure 1.

Two salivary duct carcinomas with A, B) Chromosome 7 polysomy and C,D) HER-2 amplification. (A, C) Hematoxylin and eosin sections (10x) of ductal growth pattern; (B) Strong EGFR membranous expression by immunohistochemistry (IHC); (B inset) FISH evaluation: Polysomy of Chromosome 7, green probe, with increased number per nuclei; EGFR gene, red probe, with similar increased number as chromosomal probes (on average 4 copies per nucleus); DAPI stain highlighting nuclei (blue). (D) Strong complete HER-2 membranous expression by immunohistochemistry (IHC); (D inset) FISH evaluation: chromosome 17, green probe, generally showing two copies per nucleus; HER-2 gene amplification, red probe, showing clusters of probe corresponding to marked gene amplification; DAPI stain highlighting nuclei (blue).

Figure 2.

Kaplan Meier curve of overall survival of salivary duct carcinoma patients by the average number of copies of chromosome 7.

Figure 3.

Kaplan Meier curve of overall survival of salivary duct carcinoma patients by categorical classification based on the number of copies of chromosome 7.

Chr 7 polysomy was present in 13 (28.3%) of the 46 tumors with EGFR expression (Table 1). Specifically, polysomy was noted in 2 (11.8%) of 0+ tumors; 2 (16.7%) (1+); 5 (31.3%) (2+) and 6 (40.0%) (3+ tumors) although this association did not quite reach statistical significance (p=0.06).

d) Mutational analysis

Our mutational analysis, the first in these tumors showed that five (9.4%) of the 53 tumor specimens suitable for this analysis had mutations in exons 18 and 19, hot spots of the EGFR gene: three in exon 18 and two in exon 19 (Supplementary Figure 1). Point mutations in exon 18 were identified in codons L688P, A698T and L718P. A 15 base pair in-frame deletion, E746_A750del, and a point mutation in codon S752P were identified in exon 19. No mutations were identified in Exon 21 (Table 3). EGFR expression by IHC was present in four of the five mutated tumors; two, 3+, one, 2+ and one, 1+ expression. Polysomy of Chr 7 was present in one of the three cases evaluable by FISH. HER-2 was not expressed or amplified in any of these five tumors. All five patients presented with advanced disease (stage III or IV), and 4 of the 5 died from disease from 7 to 42 months following diagnosis; all four developed lymph node or distant metastases.

Table 3.

EGFR gene mutations identified in salivary duct carcinomas.

Sample	EGFR EXON	Type of mutation	Nucleotide base change	Protein change	EGFR IHC	Chr 7 copy # average	Age	Sex	Race	Tumor Stage	Lymph Node Stage	Size (cm)	Outcome	F/U (mo)
						Polysomy
S54	18	point mutation	2063T<C	L688P*	1†	3.6	61	M	W	T4b	N2b	3	DOD	7
S22	18	point mutation	2153T<C	L718P*	1	1.7	57	F	W	T3	N0	3	L-NED	102
S36	18	point mutation	2092G<A	A698T	0	na	73	M	W	T4a	N2b	4	DOD	9
S8	19	micro-deletion	2236_2250del	E746_A750del*	3	na	41	M	W	T3	N2b	2	DOD	13
S4	19	point mutation	2254T<C	S752P	3	2.1	58	F	W	T4a	N0	>4.0	DOD	42

^*mutations also identified in lung adenocarcinomas

^†3+ EGFR expression on whole tissue section

All tumors arose in the parotid.

Chr, chromosome; DOD, dead of disease; F, female; F/U, follow-up interval; IHC, immunohistochemical expression; L-NED, living no evidence of disease; M, male; mo, month; na, not available; W, white.

HER-2

a) Immunohistochemistry (IHC)

Seventeen (25.8%) of the 66 salivary duct carcinomas were positive for membranous HER-2 staining (Table 1). Of these, strong continuous membrane expression (3+) was present in ten (58.8%); weak continuous staining (2+) in three (17.6%), and weak partial staining in four (23.5%).

No correlation between HER-2 expression levels and clinical factors including age, sex, T and N stage or clinical outcome were found (Supplementary Table 1).

b) Gene Amplification (FISH)

HER-2 gene amplification by FISH was present in 8 of the 66 (12.1%) tumors. Seven of the 8 amplified tumors showed protein expression; six (66.7%) with strong immunostaining (3+) and 1 with weak staining (1+) (Table 1). The gene amplified tumor with negative IHC expression on tissue array showed areas of strong, (3+) expression on the corresponding whole tissue section (heterogeneous distribution). HER-2 gene amplification was characterized by an excess of five fold (10+ copy number) per cell showing clusters of probe signal (Figure 1D). There was a significantly higher proportion of gene amplified cases in the 3+ IHC expression group than the tumors with low expression (p=0.0004).

c) Chromosomal Ploidy

Chr 17 polysomy, an average of >2.5 copies per cell, was present in eight (15.7%) of 51 tumors; of these eight, two were HER-2 positive by IHC (one, 3+ and one, 1+) and one had HER-2 gene amplification. Ploidy classification showed 23 (45.1%) tumors with Chr 17disomy, 10 (19.6%) low trisomy, one (2.0%) high trisomy, 12 (23.5%) as low polysomy, and 5 (9.8%) high polysomy (Table 2). No correlations between Chr 17 polysomy and IHC expression, gene amplification, age, sex, T and N stage or clinical outcome were noted regardless of polysomy categorization (Supplementary Table 1).

Correlations between HER-2 and EGFR

Similar expression profiles of EGFR and HER-2 were present in 22 (33.3%) of tumors (5, 3+; 2, 2+; 0 1+, and 17, 0+). Three of the eight HER-2 amplified tumors also had Chr 7 (EGFR) polysomy (> 2.5 average copy #). Polysomy of chromosomes 7 and 17 was found in tumors with different levels of EGFR and HER-2 protein expression (Supplementary Table 2). No positive correlation between EGFR and HER-2 alterations was found (p=0.008) suggesting independent roles for these markers in salivary duct carcinoma.

Discussion

Our concurrent analysis, the most comprehensive thus far, of the expression and the genomic alterations of the EGFR and HER-2 receptors revealed shared and variable manifestation of these genes in salivary duct carcinomas. In addition, the mutational (the first in this entity), expression, and amplification screening together with Chr 7 ploidy analysis provides broad genomic and phenotypic account of the EGFR alterations in this entity. The results show a trend for association between high EGFR expression and Chr 7 polysomy and poor clinical outcome, but this did not reach a statistical significance (29, 32). In this study, the lack of correlation between EGFR gene amplification and protein expression suggests that alternative mechanisms including post-translational modification, or decreased degradation may be involved in the upregulation of this gene in salivary duct carcinoma (33, 34). Up-regulation of EGFR gene transcription through regulatory proteins and transcription factors (33) cross-signaling with other growth factors and via hormonal pathways have been suggested (34, 35).

The detection of five tumors with mutations in the EGFR gene in Exons 18 and 19, suggests tyrosine kinase inhibitors (TKIs), such as erlotinib may indeed provided targeted therapeutic options for a subset of salivary duct carcinoma patients. Several of these activating mutations were identified at sites that have previously correlated with high response rates to TKIs, gefitinib or erlotinib in lung adenocarcinomas (21-23). We, however, noted that cases with mutations manifested variable levels of EGFR expression. This finding as previously noted in lung carcinomas suggests IHC is a poor surrogate for mutational status/clinical response to TKIs (9, 36-38). Therefore, molecular testing of the EGFR gene sequence is recommended to identify the subset of patients likely to benefit from these targeted TKIs like erlotinib. Moreover, we identified Chr 7 polysomy in a quarter of salivary duct carcinomas. Recent studies in lung and colonic carcinomas have suggested high polysomy for Chr 7 may also predict responsiveness to TKIs perhaps by reducing the rate of resistance to such therapies (25, 38). Thus as more correlative information becomes available, molecular assessment through FISH analysis may provide a predictive assessment for therapeutic response.

Our results also show significant survival differences between patients with and without Chr 7 polysomy (Figure 2). Although, the rate limiting polysomy number of biological significance is not known, our analysis of categorical groupings showed that even low levels of chromosomal duplication affected rate of progression to death (Table 2, Figure 3). Similar to other carcinomas, polysomy/EGFR copy number in our study correlated with poor prognosis suggesting DNA aneuploidy and/or relative increase in EGFR copy number plays an adverse biological role (14, 24, 26-28). Interestingly, previous reports have shown that response to cetuximab and panitumumab, two epidermal growth factor inhibitors (tyrosine kinase inhibitors) correlated with EGFR copy number (polysomy and amplification) in colonic adenocarcinoma (25) and not mutational status. Similar correlation was noted in lung carcinomas, with response to gefitinib though EGFR mutations and HER-2 amplification were also frequently present in this subgroup of patients (31, 39). The low level amplification (> 2.5 chromosomal copies per cells) found in this study suggests that even a small increase in gene dose may be clinically significant and need to be targeted by a therapeutic agent.

Heterodimerization of EGFR and HER-2 growth factor receptors may also cooperate to promote tumor growth and progression and provide a rationale for the use of therapeutic agents or agents with duel targets (34, 40). As 20% of salivary duct carcinomas express combined EGF and HER-2 receptors, identification of this population may be required for more directed therapy. Moreover, lapatinib ditosylate, a small molecule inhibitor of both EGFR and HER-2 kinase activity, has shown response in breast adenocarcinoma, whereas gefitinib which only blocks EGFR has not improved response in that patient population (10). These results suggest that blocking multiple members of the growth factor receptor pathway may lead to better reponse in a subset of these patients. Such complex interactions of the EGFR pathway and targeted agents will require further investigations with careful correlation to uncover the mechanisms leading to therapeutic response.

Our results support those of others that HER-2 amplification is present predominately in tumors with high (3+) expression (41, 42) and appears to be the dominant mechanism for HER-2 over-expression in this tumor type. This finding suggests that immunohistochemistry evaluation may reflect the functional status of this gene in most salivary duct carcinomas though amplification is notably lower (60-80%) than breast adenocarcinomas (90%) with high (3+) HER-2 expression (7, 41-43). Moreover, the incidence of HER-2 expression by IHC in salivary duct carcinomas is similar to breast adenocarcinoma (15). Our findings are consistent with those of Jeahne et al.(8) whereby the HER-2 high (3+) expression in the 50 salivary duct carcinomas in that cohort was 20.6% similar to ours and others (3), though is notably lower than in smaller reports (41, 44) which may reflect grouping of 2+ in the high expression category and tumor selection by immunoexpression in some studies. It is worth noting, however, that the incidence of HER-2 expression was slightly lower in this study (15.2%, 3+) than in our previous report that included a majority of these tumors (3). The difference can be attributed to intratumoral heterogeneity (18, 45) of HER-2 and utilization of microarray sections which underscores that certain markers could be underestimated using tissue array or on small biopsy specimens.

HER-2 gene amplification in a subset of these tumors highlights the potential therapeutic use of trastuzumab (Herceptin) in the management of selected patients similar to the treatment in mammary carcinoma (46). Trastuzumab, a humanized monoclonal antibody, is directed against the extracellular domain of the HER-2 tyrosine kinase receptor. Identifying the subset of patients who may benefit from trastuzumab is important to target the patients who may benefit from treatment (47). This is further supported by initial evidence of response of salivary duct carcinoma to trastuzumab (14, 48, 49).

Conclusion

Our results show that molecular alterations of the EGF family of growth receptors now known to be indicators (possible biomarkers) for potential targeted therapy are present in a subset of salivary duct carcinomas. Both activating mutations (10%) in the EGFR gene and Chr 7 polysomy (25%) were identified in a subset of salivary duct carcinomas. These alterations have correlated with tyrosine kinase inhibitor treatment response in different adenocarcinomas and may potentially be applicable in salivary duct carcinoma patients. Additionally Chr 7 polysomy portends a poorer clinical outcome which trends with high EGFR expression. The finding of both over-expression of HER-2 and gene amplification defines a group of patients who may benefit from targeted therapy with trastuzumab. The results of this study highlight the spectrum of alterations in the EGFR family of receptors as potential targets for therapy in salivary duct carcinomas.

Supplementary Material

1

Supplementary Figure 1. Examples of salivary duct carcinomas with EGFR gene mutations: (A) An exon 18, point mutation (the native codon is designated followed by an arrow showing the altered codon sequence and the resulting amino acid alteration and location within the gene) and (B) a 15 base pair in-frame deletion in exon 19 with the location of the deleted amino acids.

Statement of Translational Relevance.

Our concurrent analysis, the most comprehensive to date, of the expression and the genomic alterations of the EGFR and HER-2 receptors define the molecular alterations of these genes in salivary duct carcinomas for the biological stratification of patients with these tumors. Both activating mutations (10%) in the EGFR gene and Chr 7 polysomy (25%) were identified in a subset of salivary duct carcinomas. These alterations have correlated with tyrosine kinase inhibitor treatment response in different adenocarcinomas and may potentially be applicable in salivary duct carcinoma patients. Additionally Chr 7 polysomy portends a poorer clinical outcome which trends with high EGFR expression. The finding of both over-expression of HER-2 and gene amplification defines a group of patients who may benefit from targeted therapy with trastuzumab. The results of this study highlight the spectrum of alterations in the EGFR family of receptors as potential targets for therapy in salivary duct carcinomas.